Heparan sulfate binds many growth factors, chemokines, morphogens, extracellular matrix proteins, enzymes and enzyme inhibitors via relatively short sulfated saccharide sequences. One relatively rare modification to the chains, the addition of sulfate to carbon-3 of glucosamine residues, is poorly understood in terms of its biosynthesis and function. A family of seven glucosaminyl 3-O-sulfotransferases (Hs3st) exists in vertebrates. Although the enzymes have been cloned and partially characterized, little is known about the context in which these modifications occur and even less is known about the endogenous ligands that bind to heparan sulfate chains that contain 3-O-sulfate groups. The central hypothesis of this proposal is that the Hs3sts act selectively on subsequences in heparan sulfate and thereby generate specific binding sites for endogenous protein ligands. This proposal focuses on (i) methods to structurally characterize 3-O-sulfated heparan sulfate, (ii) the specificity of Hs3st-1 and Hs3st-2 as model 3-O-sulfotransferases, (iii) the discovery and characterization of natural ligands that bind to Hs3st modified chains, and (iv) examination of heparan sulfate derived from mice altered in Hs3st-1 and Hs3st-2. Towards these goals, we have the following specific aims: Aim 1: Develop a quantitative method for determining 3-O-sulfation of disaccharide and tetrasaccharide subunits in heparan sulfate. Aim 2: Generate functionally active 3-O-sulfated heparan sulfate using Hs3st-1 and Hs3st-2. Aim 3: Capture and identify protein ligands that bind to 3-O-sulfated heparan sulfate. Aim 4: Examine the formation of binding sites by Hs3st-1 and Hs3st-2 in vivo. We expect that this project will significantly enhance our understanding on how this class of sulfotransferases influences biological processes and pathological states, and validate them as potential targets for pharmacological intervention.